A Three-Step Kinetic Mechanism for Peptide Binding to MHC Class II Proteins

Abstract

Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M -1 s -1 ) and dissociation (<10 -5 s -1 ) processes. Various linear and branched pathways have been proposed to account for these data. Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins. Our experiments identified an obligate intermediate in the binding reaction. The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change. The same mechanism is observed for different peptide antigens. In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding. The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed.Proteins encoded by the major histocompatibility complex (MHC) 1 gene locus bind peptide antigens and display them at the cell surface for inspection by the immune system as part of the mechanism by which foreign material in the body is recognized and removed (1). Class II MHC proteins generally are found on specialized immune system cells such as B cells, macrophages, and dendritic cells, but they can be expressed by most cell types in response to inflammation or infection (2). Newly synthesized class II MHC R-and -glycoprotein subunits associate with a chaperonin-like invariant chain protein, which places an extended loop in the class II peptide binding site and directs transport to an endosomal compartment (3). Endosomal proteins cleave the invariant chain, and the bound fragment is exchanged for peptides generated from cell-surface and endocytosed proteins, in a poorly characterized process catalyzed by the peptide-exchange factor HLA-DM (4). MHC-peptide complexes then are transported to the cell surface for inspection by T-cell receptors on CD4 + T lymphocytes. Crystal structures have been determined for several human and murine class II MHC proteins in complex with defined peptides (5-13). In each case, the peptide was bound in a polyproline type II-like conformation, with several side chains projecting into specificity-determining pockets within the overall peptide binding groove, and with many additional contacts between the MHC proteins and the main chain of the bound peptide.Initially, in vitro kinetic measurements of peptide binding to purified class II MHC proteins were interpreted in terms of the simple bimolecular reaction shown in Scheme 1 (14): Physical and chemical characterization of purified class II MHC revealed that they carried complex mixtures of tightly bound endogenous peptides (15-19). The stoichiometry of binding for peptide added to these preparations was quite low. Many of the natural peptide ligands had extremely long half-lives, often on the order of ...

Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide

“…In general, such processes are characterized by funnel-shaped energy landscapes, with a large number of less stable conformers and fewer low energy forms. Viewed in this light, DM appears to be recognizing and editing out rare MHCII-peptide conformations that retain some mobility characteristic of intermediates in the overall multistep MHCII-peptide binding process (72).…”

Cooperativity of Hydrophobic Anchor Interactions: Evidence for Epitope Selection by MHC Class II as a Folding Process

“…Such approaches fail to account for the evidence that significant conformational rearrangements occur in the peptide/ MHCII complex during binding. Indeed, detailed kinetic analyses of peptide binding to empty MHCII or to a fast dissociating peptide/MHCII complex, have pointed out the presence of two distinct conformers of MHCII: an open or fast dissociating species and a closed kinetically stable form (9,10). Moreover, it has been known for some time that MHCII molecules must bind to a peptide for efficient cellular trafficking and presentation and to prevent aggregation of the empty form (11).…”

Small Molecules That Enhance the Catalytic Efficiency of HLA-DM

“…1C) (32,33). A real-time peptide binding assay involving fluorescence resonance energy transfer from tryptophan residues of the MHC to a fluorophore attached to the peptide has also been reported (34). An advantage of the FP method is that it does not require excitation in the UV range, conditions under which many small molecules are highly fluorescent.…”